With a concern about depletion for fossil fuels, alternative fuels are now being developed. In particular, bioethanol derived from biomass is focused because biomass is a renewable resource which occurs in great abundance on earth, and can be used without increasing carbon dioxide in the atmosphere (carbon neutral) to contribute to prevention of global warming.
However, currently, mainly corn and sugar cane are used as raw materials to produce bioethanol, which causes competition with food. Therefore, it is desired in the future to produce bioethanol using lignocellulose-based biomass, such as rice straw, straw, and wood scrap, as a raw material to avoid the competition with food.
Lignocellulose-based biomass is composed mainly of three components, cellulose, hemicellulose, and lignin. Among these, cellulose can be converted to glucose by saccharification, and then used in ethanol fermentation by a glucose-utilizing yeast such as Saccharomyces cerevisiae or the like. In contrast, hemicellulose can be converted to a pentose such as xylose or arabinose by saccharification, but is hardly used in ethanol production by fermentation in that naturally-occurring yeasts have a very poor ability to utilize xylose or arabinose.
Accordingly, for xylose utilization, a yeast has been genetically engineered to overexpress xylose reductase (XR) and xylitol dehydrogenase (XDH) derived from the yeast Pichia stipitis and xylulokinase (XK) derived from the yeast Saccharomyces cerevisiae by introducing the genes for these enzymes into the yeast (Non-Patent Documents 1 and 2). In addition, a yeast that allows ethanol fermentation from xylose has been made by introducing genes for xylose isomerase (XI) derived from anaerobic fungus Piromyces or Orpinomyces and XK derived from the yeast Saccharomyces cerevisiae into the yeast to express them (Non-Patent Document 3).
Thus, ethanol fermentation from xylose has been made possible by the creation of such xylose-utilizing yeasts. However, there are various problems with developing ethanol fermentation from xylose to an industrial scale, including, for example, a lower utilization (consumption) rate, a lower ethanol production rate, and a lower ethanol yield with xylose than with glucose; and the presence of fermentation inhibitors in a saccharified biomass, which is the problem to be mostly solved for putting ethanol production from cellulose-based biomass into practical use.
Cellulose-based biomass can be degraded (saccharified) to C6 sugar such as glucose, or C5 sugar such as xylose or arabinose using the process such as enzymatic treatment, treatment with diluted sulfuric acid, or hydrothermal treatment. According to enzymatic treatment, enzymes are required in a large variety and amount, which causes the problem of cost with the development to an industrial scale; while according to treatment with diluted sulfuric acid or hydrothermal treatment, various overdegraded products (by-products) may occur, including weak acids such as acetic acid and formic acid; aldehydes such as furfural and hydroxymethylfurfural (HMO; and phenols including vanillin, and it has been known that such by-products are fermentation inhibitors which greatly inhibit ethanol fermentation from xylose (Non-Patent Documents 4 to 6). Therefore, a yeast that is tolerant to overdegraded products of biomass, or a yeast that is capable of efficient ethanol fermentation even in the presence of such fermentation inhibitors is desired so that cost-effective procedures, treatment with sulfuric acid and hydrothermal treatment can be used to put ethanol fermentation from biomass into practical use.
Heretofore, the influence of fermentation inhibitors on yeasts has been investigated (Non-Patent Documents 4 to 6). It has been found that furfural has a great influence on the survival, growth rate, budding, ethanol yield, biomass yield, and enzymatic activity of yeasts. It has been found that HMF causes accumulation of lipids, reduces the protein content, and inhibits alcohol dehydrogenase, aldehyde dehydrogenase, and pyruvate dehydrogenase in yeast cells. Research has been carried out using screening of disrupted strains or transcriptional analysis to search for a gene tolerant to furfural or HMF (Non-Patent Documents 7 and 8).
Meanwhile, it was thought that weak acids such as acetic acid and formic acid would affect the pH in yeast cells, in other words, weak acids would occur in the medium in an undissociated form, and the undissociated weak acid would penetrate through the cell membrane of yeast into the cytosol of the yeast with around neutral pH, and then become dissociated into an anion and a proton to cause pH decrease in the cell of the yeast (Non-Patent Document 4). Then, the pH decrease in the cell would activate ATPase to maintain homeostasis, so requiring ATP. Under anaerobic conditions, ATP is regenerated through ethanol fermentation. It seems that regarding ethanol fermentation from glucose, ATP is generally regenerated even in the presence of acetic acid without affecting the fermentation ability so much; however, regarding ethanol fermentation from xylose, ATP is poorly regenerated in the presence of acetic acid in that the fermenting ability deteriorates.
The inventors have investigated the relation between acetic acid and pH in a fermentation medium using the engineered Saccharomyces cerevisiae MN8140X strain into which the genes for XR, XDH, and XK had been introduced, and found that inhibition of fermentation does not occur in this yeast even in the presence of acetic acid when the pH is adjusted from acidic toward neutral. It has been also reported that the same results are obtained in the engineered yeast into which the genes for XI and XK have been introduced (Non-Patent Document 9).
However, the control of pH is not practical to develop ethanol production from cellulose-based biomass to an industrial scale because it is costly and the contamination with other microorganisms may occur with around neutral pH. Accordingly, efficient ethanol fermentation from xylose in the presence of acetic acid (at acidic pH) is desired.
The inventors have conducted a study of efficient ethanol fermentation from xylose even in the presence of acetic acid by the use of a xylose-utilizing yeast transformed so as to overexpress a gene for at least one of pentose phosphate pathway metabolic enzymes such as transaldolase (TAL) and transketolase (TKL) (Patent Document 1).
The inventors have also conducted a study of efficient ethanol fermentation from xylose even in the presence of formic acid by the use of a xylose-utilizing yeast transformed so as to overexpress a gene for formate dehydrogenase (Patent Document 2).
Meanwhile, further improvements in technology are desired that are suited for ethanol fermentation from xylose using an actual saccharified biomass, which contains various fermentation inhibitors.